The hepatitis C virus (HCV) is now recognized as the major agent of chronic hepatitis and liver disease worldwide. It has been estimated that HCV infects about 400 million people worldwide, corresponding to more than 3% of the world population. In the United States alone, the economic burden resulting from HCV infections is predicted to reach many billions of dollars in direct medical costs. HCV infection results in chronic infection in between 70 and 80% of cases, which often progresses to liver cirrhosis, liver failure and/or hepatocellular carcinoma.
HCV is a small enveloped flavivirus, which contains a positive-stranded RNA genome of about 10 kilobases. The genome has a single uninterrupted ORF that encodes a polyprotein of 3010-3011 amino acids in length. The structural proteins of HCV include a core protein (C), which is highly immunogenic, as well as two envelope proteins (E1 and E2), and four non-structural proteins NS2, NS3, NS-4 and NS5. It is known that the NS3 region of the virus is important for post-translational processing of the polyprotein into individual proteins, and that the NS5 region encodes an RNA-dependant RNA polymerase.
Treatment options for HCV infected individuals have significantly improved in recent years with the availability of two therapeutics, a pegylated interferon-α and the nucleoside analogue ribavirin6-9. By itself, ribavirin has little effect on HCV, but co-administration with interferon increases the sustained response rate by two- to three-fold. For these reasons, combination therapy is now recommended for hepatitis C, and interferon monotherapy is applied only when there are specific reasons not to use ribavirin.
Unfortunately, these therapies are expensive and require sophisticated medical management, factors which put this option out of reach for the vast majority of infected individuals. Accordingly, the development of a prophylactic vaccine to prevent the continued spread of HCV infection remains critical. Despite over 20 years of vaccine research in numerous laboratories around the world, there is still no HCV vaccine available today.
Nevertheless, several findings indicate that immunological control of HCV is possible. In both humans and chimpanzees, spontaneous eradication of HCV can be achieved during acute infection10-14. In addition, re-infection after initial clearance has been shown to lead to a shorter duration and lower peak viremia in the chimpanzee model15; this was also correlated with a resurgence of preexisting HCV-specific cytotoxic T-lymphocytes (CTL). Also, injection drug users with ongoing exposure who previously cleared HCV spontaneously were shown to be less likely to develop chronic HCV infection compared to those with no previous infection16.
Since the isolation of HCV in 198817, a number of studies have dissected the roles of different components of protective immunity to HCV18. While the function of humoral immune responses against HCV antigens in viral clearance and protection against reinfection is controversial, that of cellular immune responses to HCV is better understood. Virus-specific T lymphocytes, along with neutralizing antibodies, are the principal antiviral immune defense in established viral infections. Control of acute viral replication is clearly associated with expansion of CD4+ and CD8+ T cells18. Whereas CD8+ cytotoxic T cells eliminate virus-infected-cells, CD4+ T cells are essential for the efficient regulation of the antiviral immune response. CD4+ T cells recognize specific antigens as peptides bound to autologous HLA class II molecules. Several observations support an important role of CD4+ T cells in the elimination of HCV infection (Tsai et al., 1997, Hepatology 25: 449-458; Diepolder et al., 1995, Lancet 346: 1006-1009; Diepolder et al., 1997, J. Virol. 71: 6011). In a cohort of patients 20 years after exposure to HCV, about 40% of recovered patients had no detectible antibody response, whereas HCV-specific helper and CTL responses persisted19. HCV-specific neutralizing antibodies were readily detected in patients with chronic infection and impaired virus-specific CD4+ T-cell response, but not in patients who cleared infection with robust virus-specific CD4+ T-cell response20. Vigorous, multispecific CTL and T-helper immune responses against HCV antigens in early infection correlate with clearance of HCV infection10, 13, 21-26 and HCV-specific CTLs can persist for years after infection10, 23. Furthermore, recurrence of HCV viremia has been shown to follow the loss of HCV-specific T Helper cell responses11. In chimpanzees serially infected by HCV, recovered animals did not have detectable antibody against the HCV envelope glycoproteins, while rapid control of secondary infection was associated with a strong T-cell proliferative response and expansion of memory CD4+ and CD8+ T cells27-29.
Development of an effective HCV vaccine faces strong obstacles, principal among them being the high level of HCV protein sequence diversity30. For vaccines to be effective, responses covering a wide range of variants are required, as single amino acid substitutions can completely abrogate recognition by HLA class I restricted CD8+ cytotoxic and class II restricted CD4+ T-helper cells24.
For HIV-1, a similarly variable virus, there has been strong interest in rational vaccine design, a relatively new discipline that attempts to define optimal vaccine sequences that minimize differences from the circulating strains while maximizing immunogenicity33. Even though these vaccine constructs are in a sense artificial, HIV-1 experiments showed that artificially created consensus and ancestral envelope proteins retained folding and conformational antibody binding characteristics, and responses to the vaccine showed enhanced B- and T-cell cross-reactivity compared to natural strains34, 35.
Recently, a new computational approach to the design of polyvalent vaccine antigens for T-cell based vaccines was developed and applied to HIV-132. These antigens consist of sets of “mosaic” proteins which are computationally generated recombinants assembled from fragments of natural sequences and selected to be optimal using a genetic algorithm. Mosaic proteins resemble natural proteins, but are optimized to maximize the coverage of potential T-cell epitopes (nonamer peptides) found in natural sequences and to reduce the number of rare or unique 9-mers to avoid vaccine-specific responses. A small set of 3-4 such HIV-1 “mosaic proteins” provided comparable coverage to thousands of separate peptides. Mosaic proteins can be synthesized and expressed and are immunogenic in mice36. When mosaic constructs for HIV-1 were compared to natural strains in DNA vaccines in mice37, the two- or three-mosaic Env sets elicited the optimal CD4 and CD8 responses. These responses were most evident in CD8 T cells; the three-mosaic set elicited responses to an average of eight peptide pools, compared to two pools for a set of three natural Envs, indicating that synthetic mosaic antigens can induce T-cell responses with expanded breadth and may facilitate the development of effective T-cell-based vaccines.